Field of the Invention The invention relates to synergistic combinations of selected repellent compounds having greater pest repellent activity than would be observed for the individual compounds. The invention extends to devices comprising said compositions and the use of said compositions in repelling and controlling pests, in particular insects and/or haematophagous arthropod parasites, and most notably mosquitoes.

Background of the Invention

Invertebrates and other pests are common vectors for pathogenic organisms and are responsible for a variety of human diseases. Arthropods, including insects and ticks, account for over 80% of all known animal species, and they are one of the most important disease vectors. Bacterial, viral, nematode and protozoan diseases are spread by these vectors. Transmission of a communicable disease from an infected host insect to a conspecific individual commonly occurs either via bite, such as in the case of haematophagous pests, in particular insects or ticks, or via contaminated faeces.

One of the most commonly known insect-borne diseases is malaria which is transmitted via the Anopheles genus of mosquito. Other common mosquito- borne diseases include Yellow Fever, Dengue Fever, West Nile Virus Disease, Chikungunya, Zika and Encephalitis. Mosquitos are estimated to transmit disease to more than 700 million people annually in Africa, South America, Central America, Mexico and much of Asia. Globally, 216 million cases of Malaria occur annually and it is responsible for the death of over 655,000 people annually. Indeed, it is estimated that 6.5% of the world’s population are at risk of infection. Dengue fever, vectored by mosquitoes of the Aedes genus, is one of the world's most rapidly re-emerging diseases. Recent estimates suggest a 30-fold increase in prevalence over the last 50 years, with 96 million new cases developing each year. Aedes aegypti and particularly Aedes albopictus, the two species most responsible for the disease, have undergone rapid territorial expansion with their range now greater than when control began. Reports of insecticidal resistance are also increasingly common.

Ticks are hematophagous ectoparasites of humans and animals with a worldwide distribution. They are vectors of disparate pathogens, including protozoa, bacteria including rickettsiae and viruses, which pose a serious threat to human and animal health. The sheep tick, Ixodes ricinus, the most abundant tick in Europe, is the vector of a large variety of pathogens of medical and veterinary importance, including Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia divergens and viruses such as tick-borne encephalitis and Louping ill.

Consequently, there is a continued interest in developing control methods aimed at reducing or eradicating the incidence of vector-borne pathogenic diseases. This is often achieved by targeting the vector commonly associated with transmission. Methods for controlling infestations and infection by insects and/or haematophagous parasites have typically been in the form of; physical barriers preventing transmission; chemical compositions, such as insecticides, biopesticides including all natural or biological pesticides such as entomopathogenic fungi and semiochemicals, chemical drugs and repellents; and also biological controls such as immunisation.

Increased use of insecticide-treated nets (ITN), usually bed nets, and indoor residual spraying (IRS) has been successful in reducing the malaria burden of many African and Asian countries. Typically, pyrethroid insecticides are used in the nets and for the spraying. However, there is concern that over dependency on the pyrethroid class of insecticides will lead to wide-scale selection of pyrethroid resistant insects thus deleteriously impacting the future control of malaria and other mosquito-transmitted diseases. In fact, the negative impact of pyrethroid resistance on the effectiveness of ITN against malaria has already been demonstrated in hut and household trials.

However, as technology developed, an increasing amount of research has begun to focus on the development of products attained from natural products, especially in the wake of increasing concerns for human health and the environment with regards to the use of chemical pesticides not to mention increasing reports of resistance to established repellents such as N,N-Diethyl- meta-toluamide (DEET), the most common active agent in insect repellents. A large proportion of research conducted to find alternate sources of repellency focuses on essential oils, extracted from plants often used in traditional senses. In most of the studies conducted, it has been found that the repellent effects of such essential oils have increased in accordance with increases in the concentrations of the oils concerned meaning that to form effective repellents large quantities of oil must be extracted. Further, inconsistencies in the ability of any organism’s volatile bouquet to elicit a behavioral response in insects often lies in their chemical profile; as the active chemicals causing the repellency may only be present in small amounts, being far outweighed by less effective compounds or even those that elicit an opposite response.

Therefore, there remains a need for enhanced efficacy pest, in particular insect and/or haematophagous parasite, repellant formulations, and preferably high efficacy insect repellant formulations comprising active compounds that can be isolated and synthesized without having to process swathes of plant material. Thus, there is an environmental and economic need for such high efficacy insect repellant combinations containing lower levels of individual repellant compounds.

The inventors have discovered a discrete group of compounds, which are known to be present in pine-spruce extract essential oils, that are detected by adult mosquitoes and effectively function to repel them from the source of said compounds. Notably, when certain of these compounds were present in combination, it was unexpectedly found that said compounds interact synergistically to impart enhanced repellent efficacy, and therefore these combinations represent novel and synergistic repellent compositions for use in reducing insect, in particular mosquito, populations and incidence of associated pathogen transmission. Further, these combinations of compounds were also shown to have a potent repellent effect on other pests, notably haematophagous parasites such as ticks.

Statements of Invention

The present invention, in its various aspects, is as set out in the accompanying claims.

During the development of the compositions of the present invention, the inventors identified 10 compounds that are found in essential oil extracts of pine and spruce species of the Pinaceae family, which individually act as mosquito repellent semiochemicals; Borneol, Bornyl acetate, 2-cyclohexen-1- ol, a-pinene, b-pinene, camphor, cineole, caryophyllene, eugenol and isoeugenol. Notably, borneol, bornyl acetate, isoeugenol, eugenol, camphor and caryophyllene were found to be particularly effective achieving greater than 50% repellency when applied at a concentration of 1 mg/cm ² applied to a set surface area.

Further, blended compositions comprising of borneol, bornyl acetate, isoeugenol and eugenol as the insect repellant active component, were unexpectedly found to be synergistic in that said blends provide an equivalent or enhanced insect repellant efficacy at a combined active ingredient level lower than that of the individual repellant compounds.

Additionally, these blended compositions were also found to have a potent repellent effect on other haematophagous parasites pests, in particular ticks. Therefore, according to a first aspect of the invention there is provided a composition comprising a pest repellent component and optionally at least one carrier or diluent, wherein the pest repellent component comprises, and preferably consists of, two or more compounds selected from: borneol, bornyl acetate, isoeugenol and eugenol.

As the skilled reader will readily understand, some of the compounds of the repellent component disclosed herein include one or more asymmetric carbon atom, and so may exist in multiple spatial configurations. For example, borneol and bornyl acetate each exist in two enantiomeric forms, i.e. (+)-borneol / bornyl acetate and (-)-borneol / bornyl acetate. Therefore, when used in the context of the repellent components of the present invention, reference to any compound should not be construed as limited to any specific spatial configuration, and generally includes all stereoisomers and mixtures, e.g. racemic mixtures, thereof.

In preferred embodiments, the pest repellent component comprises or consists of eugenol and borneol, eugenol and bornyl acetate, borneol and bornyl acetate, isoeugenol and bornyl acetate or eugenol and isoeugenol. Most preferably, the pest repellent component comprises or consists of eugenol and borneol, eugenol and bornyl acetate or borneol and bornyl acetate.

In a preferred embodiment, the pest repellent component comprises, and more preferably consists of, three or more compounds selected from: borneol, bornyl acetate, isoeugenol and eugenol.

In yet a further preferred embodiment, the pest repellent component comprises, and more preferably consists of, borneol, bornyl acetate, isoeugenol and eugenol.

In exemplary embodiments, borneol and/or bornyl acetate are, if present, (-)- borneol and (-)-bornyl acetate, respectively. Preferably, the pest repellent component is an insect repellent and/or a haematophagous parasite repellent. More preferably, the insect is a mosquito.

Reference herein to an insect of the mosquito generally refers to members of the family Culicadae spp. and refers to those such as those belonging to the genus Anopheles, Bironella, Chagasia, Aedeomyia, Aedes (Stegomyia), Armigeres, Ayurakitia, Borachinda, Coquillettidia, Culex, Culiseta, Deinocerites, Eretmapodites, Ficalbia, Galindomyia, Haemagogus, Heizmannia, Hodgesla, Isostomyia, Johnbelkinia, Kimia, Limatus, Lutzia, Malaya, Mansonla, Maorigoeldia, Mimomyia. Onirion, Oplfex, Othopodomyla, Psorophora, Runchomyia, Sabethes, Shannoniana, Topomyia, Toxorhynchites, Trichoprosopon, Tripteroides, Udaya, Uranotaenia, Verrallina.

More ideally still, said insect refers to members of the Aedes, Culex, and Anopheles genus such as but not limited to, Anopheles albimanus, Anopheles arabiensis, Anopheles barberl, Anopheles Bellator, Anopheles crucians, Anopheles cruzll, Anopheles culicifacies, Anopheles darlingi, Anopheles dims, Anopheles earlei, Anopheles freeborn!, Anopheles funestus, Anopheles gambiae, Anopheles introlatus, Anopheles latens, Anopheles maculipennis, Anopheles mouchetl, Anopheles nili, Anopheles punctipennis, Anopheles quadrimaculatus, Anopheles stephensi, Anopheles subpictus, Anopheles sundaicus, Anopheles walker, Aedes aegypti, Aedes aboriginis, Aedes albopictus, Aedes atlanticus, Aedes atropalpus, Aedes aurifer, Aedes brelandi, Aedes bicristatus, Aedes bimaculatus, Aedes camptorhynchus, Aedes cantator, Aedes cataphylla, Aedes cinereus, Aedes clivis, Aedes cretinus, Aedes deserticola, Aedes epactius, Aedes fulvus, Aedes grossbecki, Aedes hensilli, Aedes hesperonotlus, Aedes infirmatus, Aedes Intrudens, Aedes melanimon, Aedes mitchellae, Aedes notoscriptus, Aedes polynesiensis, Aedes sollicitans, Aedes squamiger, Aedes taeniorhynchus, Aedes vexans, Aedes vigilax, Culex annulirostris, Culex fuscocephala, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui, Culex tarsalis, Culex restuans, Culex nigripalpus, Culex salinarius, Culex univittatus, Culex neavei, Culex perexiguus, Culex pipiens, Culex antennatus, Culex quinquefasciatus, Culex (Melanoconion) vomerifer, Culex (Melanoconion) pedroim, Culex (Melanoconion) adamesi, Culex modest us.

Reference herein to a haematophagous parasite generally refers to any blood feeding arthropod, and in particular to members of the arachnida class. More ideally, said haematophagous parasite refers to members of the superorder Parasitiformes, and preferably to members of the suborder Ixodida. More ideally still, said haematophagous parasite refers to members of the Ixodidae family such as, but not limited to, the genus Africaniella, Amblyomma, Anomalohimalaya, Archaeocroton, Bothriocroton, Cosmiomma, Cornupalpatum, Compluriscutula, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Margaropus, Nosomma, Rhipicentor, Rhipicephalus or Robertsicus genus.

More ideally still, said haematophagous parasite refers to a member of the genus Ixodes, and most ideally is selected from Ixodes scapularis and Ixodes ricinus.

The structures of ten identified insect repellent compounds that are found in essential oil extracts of pine and spruce species of the Pinaceae family are set out in Table 1 , below.

Table 1 : Structures of Pine family extract active repellent compounds

As would be readily appreciated by the skilled reader, the composition may comprise, in combination with the essential pest repellent component, one or more additional compounds or agents with activities other than insect repellence, such as but not limited to at least one fragrant agent and/or chemical. As will be appreciated, in this preferred embodiment said fragrant agent and/or chemical does not contribute to the repellent properties of the composition but does impart a pleasant aroma to the insect repellent composition. Such an additive is particularly desirable, for example, in situations such as for use as air/room fresheners or deodorisers or perfumes.

Suitable carriers and/or diluents are well known in the art and include hexane, ethanol and diethyl ether, the purpose of which is to dispersal, dispensation, application, timed-release, encapsulation, microencapsulation, or the like to apply the composition as further described herein available to those of ordinary skill in the art. Carriers or diluents as contemplated herein are generally inert materials used in making different formulations of the repellent compositions of the present invention. The specific carrier used in any repellent composition depends on the particular application of the repellent composition. For example, the carrier or carrier material may be, for example, agronomically, physiologically, or pharmaceutically acceptable carriers or carrier materials. The carrier component can be a liquid or a solid material. The formulation may also comprise suitable additives, for example a preservative. In a preferred embodiment, the carrier is hexane or ethanol.

In a preferred embodiment, the composition of the present invention is provided in any form known in the art customary for repellents such as, but not limited to, a solution, an emulsion, a gel, sol-gels, extruded polymers (e.g. string, pellet) or the like.

More preferably still the composition is formulated for evaporation or release of the chemicals of the composition into the air. For example, the composition may be volatised either by exposure to ambient temperatures, by warming the composition, by air movement or by a combination thereof. Alternatively, it may be dispersed from a device which can provide either an aerosol discharge or a thermal fog discharge. As will be appreciated, in this manner the composition is applied as aqueous sprays, atomizing sprays, aerosols, and fogs with or without a carrier to treat objects or areas. When the composition evaporates or is otherwise released, it disperses into the surroundings and is detected by antennal olfaction by the insects, which are repelled away from the source of the composition. Thus, direct contact with the composition is not required, and the composition is repellent from a distance.

Yet more preferably, the composition is formulated to be sprayed on, deposited on, or impregnated into a support or a material such as net, fabrics, cloth, tent, to prevent insects reaching their target.

Alternatively, the composition is formulated for topical application to the skin. For example, the composition comprising the pest repellent component may be made up into a cream, ointment, jelly, solution or suspension etc. Cream or ointment formulations that may be used for the pest repellent composition are conventional formulations well known in the art, for example, as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

In a preferred embodiment of this first aspect of the invention, said composition is used with at least one other pest, preferably insect, control measure, such as but not limited to, insect residual spraying, bed nets with or without pesticides / insecticides. Without wishing to be bound by theory, it is expected that, when used in conjunction/combination with other insect control measures, especially insecticidal or trap measures, owing to the superior repellency of the claimed composition one can effect increase in mortality or trap of the insect by repelling them toward said other insect control measure. As will be known to those skilled in the art, such a strategy is termed ‘Push-pull’ and involves use of behaviour-modifying stimuli i.e. repellency (push) of the claimed composition to increase the effective trap/mortality of the insect by driving them toward the alternative insect control measure, for example, the simultaneous attracted (pull), using highly apparent and attractive stimuli, to other areas such as traps where they are concentrated, facilitating their control.

According to a second aspect of the invention there is provided use of the composition as herein disclosed for repelling insects and/or haematophagous parasites, preferably insects and more preferably mosquitoes.

The compositions according to the first aspect of the present invention are thought to repel mosquitoes, in particular, when the composition evaporates or is otherwise released, it disperses into the surroundings and is detected by, e.g. mosquitoes, which are repelled from the source of the composition. Thus, direct contact with the composition is not required, and the composition is an efficacious repellent from a distance. According to a third aspect of the present invention there is provided a method of repelling insects and/or haematophagous parasites, preferably insects, and more preferably mosquitoes, said method comprising the step of providing a composition as disclosed herein according to the first aspect of the present invention.

Insects, particularly mosquitoes, and haematophagous parasites, particularly Ixodes ricinus, may be repelled from a desired location or site by locating the composition in the desired location or site, or by releasing said composition into the environment at said location or site. The desired location may comprise, for example, a room, region thereof or item of furniture such as a bed, or an individual wherein repellent of the insect is desired or in which the reduction or elimination of an insect presence is required. As will be appreciated by those skilled in the art, this is particularly the case in regions of the world wherein mosquito borne pathogens are apparent, and so can be used as means to reduce incidence of transmission of such pathogens.

In a preferred embodiment said method further comprises providing at least one other pest control measure, such as but not limited to, insect residual spraying, bed nets with or without pesticides / insecticides, or pest / insect trap or the like. Preferably, the pest control measure is an insect and/or haematophagous parasite control measure, and most preferably is an insect control measure.

Without wishing to be bound by theory, it is expected that, when used in conjunction/combination with other pest control measures, especially pesticidal or pest trap measures, owing to the superior repellency of the claimed composition one can effect increase in mortality or trap of the insect and/or haematophagous parasites by repelling them toward said other pest control measure. Preferably, the pesticidal and pest trap measure is an insecticide and insect trap, respectively. As will be known to those skilled in the art, such a strategy is termed ‘Push-pull’ and involves use of behaviour- modifying stimuli i.e. repellency (push) of the claimed composition to increase the effective trap/mortality of the pest by driving them toward the alternative pest control measure, for example, the simultaneous attracted (pull), using highly apparent and attractive stimuli, to other areas such as traps where they are concentrated, facilitating their control.

According to a fourth aspect of the present invention provides a device for repelling insects and/or and haematophagous parasites, preferably insects and more preferably mosquitoes, said device comprising at least one repellent element, wherein the at least one repellent element comprises the composition according to the first aspect of the present invention.

The device of the present invention is safe, non-toxic and can be manufactured at a reasonable cost. The device can be inconspicuous and simple to use and is therefore suitable for domestic use. With the low level of maintenance required, the device of the present invention is suitable for wide-scale, long term use.

The at least one repellent element preferably comprises a substrate which functions to contain and release the composition according to the first aspect of the invention. The substrate may comprise filter paper, rubber septa, fibres made of a plastic-like material, such as polyethylene, polypropylene, olefin or similar materials, laminate flakes and microcapsules.

Alternatively, said repellent element is formulated for evaporation or release of the chemicals of the composition into the air. For example, the composition may be volatised either by exposure to ambient temperatures, by warming the composition, by air movement or by a combination thereof. As will be appreciated, in this manner the composition is applied as aqueous sprays, atomizing sprays, aerosols, and fogs with or without a carrier to treat objects or areas. When the composition evaporates or is otherwise released, it disperses into the surroundings and is detected by antennal olfaction by the insects, which are repelled away from the source of the composition. Thus, direct contact with the composition is not required, and the composition is repellent from a distance.

The rate of release from all controlled release formulations will also be influenced by environmental factors such as temperature, humidity, prevailing air currents and other local micro-climate parameters. In a preferred embodiment, where evaporation or air release is utilised, repeat and timed release of the repellent composition is used.

The device preferably comprises a housing which encloses and/or supports the at least one repellent element. Preferably, the housing comprises one or more openings to enable release of the composition into the environment.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

Figure 1. Differential test-arm catches in dual-port olfactometry of plant essential oil extracts at 100% and 50% applied concentrations. Aedes aegypti responses to human odours baited with essential oils from 4 plant extracts, along with a human odour only control, and 2 commercial repellents in DEET and Citronella Oil. Data presented as mean ±SEM;

Figure 2. Mean electroantennogram (EAG) responses (normalised to paraffin oil) of Aedes aegypti to 13 pine plant VOC's. Data presented as mean ± 95 % Cl. ( ^*** =p<0.001 ; ns = not significant);

Figure 3. Differential repulsion from human odours under the presence of varied concentrations of each tested pine volatile. Aedes aegypti responses to 1 , 0.5 and 0.1 mg/cm ² of each of the electrophysiologically active pine VOC applied per set surface area. Data presented as mean value ± SEM; and

Figure 4. Differential repulsion from human odours under the presence of 0.5 mg/cm ² applied concentration of each tested pine volatile. Aedes aegypti response to each electrophysiologically active pine VOC (B - Borneol; BA - Bornyl Acetate; C - Camphor; E - Eugenol; IE - Isoeugenol) presented as mean % catch for each component, relative to ethanol pad control. Figure 5. Differential repulsion from human odours under the presence of varying concentrations of each tested pine volatile. Aedes aegypti response to each electrophysiologically active pine VOC presented as mean % catch, relative to empty cellulose sample pad control. Mean % catch for Borneol (B) & Bornyl acetate (BA) applied at 0.7mg/cm ² & 0.9mg/cm2 per unit area loaded on 30mm ² cellulose pads.

Figure 6. Repulsion from human odours under the presence of blends of pine volatile repellent compounds. Aedes aegypti response to blends comprising Borneol, Bornyl Acetate, Eugenol and Isoeugenol) presented as mean % catch for each component, relative to ethanol pad control.

Figure 7. Repulsion from human odours under the presence of two- component blends of pine volatile repellent compounds. Aedes aegypti response to blends comprising two components selected from Borneol, Bornyl Acetate, Eugenol and Isoeugenol) presented as mean % catch for each component, relative to ethanol pad control.

MATERIALS AND METHODS

Plant Materials

4 plant species were selected for initial extraction and assay: Lavendula angustifolia, Ocimum suave, Picea sitchensis & Rosmarinus officinalis. All plants were grown and maintained at Swansea University; Picea sitchensis was kept in an outdoor potted collection, while the 3 other plants were grown in a greenhouse incubated at 27±4°C. 1200g of leaf material were collected and stripped from fresh cut branches in preparation for essential oil extraction and olfactometry. Mosquitoes

Aedes aegypti (Strain: AeAe) eggs were procured from London School of Hygiene and Tropical Medicine (LSHTM). Eggs were hatched in tap water and raised using crushed guinea pig pellets (PetsAtHome own brand) through to adult stages. Adult mosquitoes were kept in bug dorms (Watkins and Doncaster) measuring 30x30x30cm and fed on a solution of 10 % sucrose solution for at least 3 days in order to fully mature. Adult females were selected for sensitivity to human odours through presentation of a human arm near the outer netting of the bug dorm. Those females that displayed positive taxis towards the arm, followed by probing behaviour, were removed and placed into a separate bug dorm, without a food source, for 24hrs prior to experimentation. None of the selected females had previously been blood fed.

Ticks Assays were conducted using /. ricinus nymphs, the most abundant developmental stage in the field and the most prevalent in disease transmission. Healthy, unfed nymphs that actively sought hosts were used in assays.

Essential Oil Extraction

Steam distillation, followed by solvent extraction, was the preferred method for essential oil extraction to ensure a consistent and pure extract in each case. 400g of stripped leaf material was coarsely broken in a blender before being placed in the biomass flask above a round-bottomed boiler flask containing 500m L distilled water. Vacuum grease was used to seal all quick-fit fixings before a vacuum was applied to the apparatus and the boiler flask was submerged in a water bath set at 63°C to initiate the distillation process. This was left to continue until all 500m L of distilled water had passed through the system and condensed in the collection flask. The 500ml_ was then separated into 100 mL samples to each of which 50ml_ of diethyl ether (99.99%; Fisher Scientific) added to each. The mix of extracted water and diethyl ether was gently agitated for 2 mins before the water was removed in a separatory funnel, this process was repeated 6 times for each 100ml_ water sample. Once all solvent extractions had been completed, the resulting diethyl ether extract was left in an open 1 L beaker suspended in a water bath at 38°C in a fume hood until all solvent had evaporated, leaving the extract that was to be used in following experiments. The process was repeated 3 times for each plant species. Each of the extracts was resuspended in 1 ml_ hexane (>99.99%; Sigma Aldrich) for experimental purposes.

Chemicals

As a potential alternative to the use of essential oil extractions as repellent formulations, chemicals known to be abundant in the volatile bouquet of members of the Pinaceae plant family were tested for repellent action. 13 chemicals were chosen in total: acetic acid, borneol, bornyl acetate, camphor, caryophyllene, cineole, 2-cyclohexen-1-ol, eugenol, eugenyl acetate, isoeugenol, a-pinene, b-pinene, a-terpineole. Repellency tests were conducted using either the (+)- and (-)- enantiomeric forms of borneol, and either a racemic mixture of (+)- / (-)-bornyl acetate or specifically (-)-bornyl acetate. No significant variation in repellent activity was observed between different enantiomers or between individual and combinations of enantiomers, indicating that activity was not limited to any particular enantiomeric form.

All chemicals were purchased from Sigma-Aldrich, Acros or Alfa Aesar unless indicated otherwise and generally had >95% purity.

Mosquito Repellence:

Olfactometry

For initial olfactometry assays, a modified y-tube olfactometer was constructed from clear Perspex, with aluminium trap doors, according to dimensions specified in Geier et al. 1999. Olfactometry assays were conducted as a modified version of host attraction-inhibition assays described in WHO 2013; whereby quantities of extract used in experimentation were reduced to 50pL in order to preserve extract, and mosquito numbers were increased to 20. In both cases, preliminary experimentation revealed no difference in responses observed between the modified protocol and that of the WHO 2013 protocol.

Initial olfactometry assays were conducted using a 50pL hexane suspension of each of the plant extracts, absorbed into filter paper measuring 20x20mm and folded every 5mm to aid evaporation. Concentrations of each extracted essential oil, along with DEET and citronella, were applied and tested at 100% & 75%. Socks, worn for a period of 24 hours prior to experimentation, were used as a host odour source throughout all experiments. A single sock was placed each test-port of the olfactometer in addition to a filter paper containing either essential oil/ repellent (treatment) or hexane only (control).

Control experiments were performed to ensure against contamination from previous experimentation through assays completed with: (i) a host odour source (sock) only in each of the olfactometer test-ports (ii) no odour source in either test-port. Between replicates, test-ports were removed from the olfactometer and cleaned using anionic surfactants. After reinstallation of clean parts, the olfactometer was left to run for 5 minutes to clear any remaining odours from the system. All experiments were performed under a fume hood using an air flow rate of 0.2m/s in the test ports and 0.4m/s in the release port. 10 replicates were completed for each experiment at each treatment dosage. Results were determined as a percentage of the total number of mosquitoes attracted to treatment ports in each experiment. Repellency was indicated through the differential percentage attraction of mosquitoes to tests ports containing both host odours and repellents, and those containing host odours and hexane only. The same protocol was used for an initial investigation into the effects elicited by specific chemicals. Chemicals were assayed as singular components, using the same basic methodology, at concentrations applied as a liquid per defined unit surface area of 1 mg/cm ² , 0.5 mg/cm ² and 0.1 mg/cm ² . Through this the most effective compounds were identified for further testing.

Further olfactometry tests were conducted using the BG-cage test to assess the effects elicited by high efficacy repellent compounds (i.e. borneol, borneol acetate, eugenol, isoeugenol and camphor), again initially as singular components and then in blended combinations. However, camphor was excluded from tested blends due to toxicity. The BG-cage test protocol was developed by Biogents (Obermayr et al., 2010) and is based on the guideline published by the American Environmental Protection Agency (EPA, 2010) in accord with WHO recommendations (WHO, 2009). In the BG-cage tests, female mosquitoes were selected that exhibited host seeking behaviour and having never received a blood meal, and 30 such mosquitoes were released into BG test cages. BG cages (41 x 41 x 16cm) have a volume of 27,000 cm ³ and a basal port with a meshed insert where a test subject hand or arm is exposed to the insects. A repellent dispenser / sample pad was placed on the hand and the efficacy of same was measured by counting the number of mosquitoes that landed and probed the mesh over a 5 minute period. Results were compared to a negative control (carrier, i.e. ethanol, without active components or empty sample pad).

Electroantennography (EAG)

Adult female Ae. aegypti were used to test the mosquito repellence of various individual compounds known to be present in the VOC profile of members of the Pinaceae plant family. Mosquitoes were 4-6 days old (post emergence), and none had blood-fed. Each chemical was assessed against three Aedes aegypti adults per replicate, and the whole experiment repeated ten times in total; giving a total of thirty individual replicates per treatment. Electroantennographical assays were carried out according to the principles set out in Abdullah et al. 2014.

Mosquito heads were excised at the pronotum. Both antennae were excised between segments 12 and 13 (distal) and both antennae were inserted into the recording electrode, while the reference electrode was inserted into the base of the excised head, just below the proximal ends of the antennae.

The glass electrodes were filled with Ringer's solution for increased sensitivity (Maddrell 1969). Authentic standards of each chemical treatment, prepared in 10pL. paraffin oil, were applied to strips of filter paper at a dose of 1mg. The filter papers were inserted into a disposable glass Pasteur pipette, which was then allowed to sit for 1 minute prior to injection; this ensured a constant release rate during injection of the VOCs in each experiment. A 5ml_ syringe was then attached to the end of the pipette which was depressed to expel the VOC's into a purified airstream at a flow rate of 1 L min-1 (20 cm/second air speed) through a glass tube (I.D= 12 mm) and over the prepared antennae. The resulting peaks were consistent against a standard of paraffin oil.

The EAG equipment consisted of a 10x gain universal probe (Syntech, Netherlands) and an IDAC 2 Signal A and an IDAC 2 Signal Acquisition Processor (Syntech). Data were analyzed with EAGPro Version 2 software, (Syntech). Paraffin oil was tested at the beginning of each experiment and used to normalise the recordings to the largest response in each set. To do this, each treatment response was calculated as a percentage of the largest response to paraffin oil in during each set (Abdullah et al. 2014), ensuring that results were comparable to each other for statistical analysis.

Those chemicals that were identified as eliciting an electrophysiological response were selected for further investigation. Single Chemical Olfactometry

Each of the chemicals to which mosquitoes were responsive, identified from the electroantennography assays, were assayed separately. Initial assays were conducted using compounds applied at 1 mg/cm ² , 0.5 mg/cm ² , and 0.1 mg/cm ² of liquid per unit area. Hexane was used to dilute each of the chemicals tested.

Further assays, using five highly repellent compounds, were carried out using the using the BG-cage test method in order to determine the minimum repellent concentration (MRC) - the minimum concentration of each individual compound required to achieve a designated threshold for repellency action, which was defined as a mean percentage mosquito trap catch of 35% or less relative to negative control. Ethanol was used to dilute each of the chemicals tested.

Multi Chemical Blends - Synergy testing

Four of the most repellent chemicals were then assayed in combinations to ascertain whether said blends provided a synergistic enhancement of mosquito repellency. Assays were conducted as per the BG-cage test protocol for all previous olfactometry experiments.

The synergism of combinations of the present invention was demonstrated by comparing the minimum applied concentration of each individual repellent compound, acting alone, that is sufficient to achieve the designated threshold for repellent action (i.e. a mean percentage mosquito trap catch of 35% or less relative to negative control) with the applied concentration of each individual repellent compound, present in a mixture, that is sufficient to achieve the same threshold repellent action.

In particular, the presence and extent of synergy was quantified for four component combinations using the ratio determined by the formula: Q ₉ /QA + CWQB + Qc/Qc + Qd/C = Synergy Index ("SI") wherein:

QA = applied concentration of compound A (first component, borneol acetate (‘BA’)), acting alone, which produced the threshold repellent action (minimum repellent concentration (MRC) of Compound A).

Q _a = minimum applied concentration of compound A, in a mixture, which produced the threshold repellent action.

QB = applied concentration of compound B (second component, (-)- borneol (‘B’)), acting alone, which produced the threshold repellent action (MRC of Compound B).

Q _b = minimum applied concentration of compound B, in a mixture, which produced the threshold repellent action.

Qc = applied concentration of compound C (third component, eugenol (Έ’)), acting alone, which produced the threshold repellent action (MRC of Compound C).

Qc = minimum applied concentration of compound C, in a mixture, which produced the threshold repellent action.

QD = applied concentration of compound D (fourth component, isoeugenol (‘IE’)), acting alone, which produced the threshold repellent action (MRC of Compound D).

Q _d = minimum applied concentration of compound D, in a mixture, which produced the threshold repellent action.

As will be readily appreciated, when the sum of Q _a /Q _A, Q _b /Q _B, Q _c /Qcand Q _d /Qp is greater than one, antagonism is indicated. Likewise, when the sum is equal to one, additivity is indicated, and when less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Similarly, the presence and extent of synergy was quantified for two component combinations using the ratio determined by modification of the above formula:

Q ₉ /QA + CWQB = Synergy Index ("SI") wherein:

Q _A = applied concentration of compound A (first component, borneol acetate (‘BA’)), acting alone, which produced the threshold repellent action (minimum repellent concentration (MRC) of Compound A).

Q _a = minimum applied concentration of compound A, in a mixture, which produced the threshold repellent action.

Q _B = applied concentration of compound B (second component, (-)- borneol (‘B’)), acting alone, which produced the threshold repellent action (MRC of Compound B).

Q _b = minimum applied concentration of compound B, in a mixture, which produced the threshold repellent action.

Statistical Analyses

Data from all olfactometry assays were analysed according to the principles set out in Geier et al. 1999. Mean (±SE) responses were calculated for each individual stimulus. The resulting mean percentage responses were then arcsine transformed to meet homogeneity assumptions. Transformed data were then analysed via ANOVA, with Tukeys post-hoc test, in order to determine differential repellency between each stimulus.

Electrophysiological responses to chemical stimuli were analysed as per the protocol in Abdullah et al. 2014. EAGPro Version 2 (Syntech) software was used to collect all EAG results. Data collected were normalised against the largest response to control stimuli in each experimental set. Resulting percentage ratios were square root transformed to obtain homogeneity of variance before being analysed via ANOVA with Tukeys post-hoc test. All statistical analyses were calculated using SPSS v22. (IBM Corporation, USA).

Tick Repellence:

Moving Object Bioassav

Tests were conducted according to the moving object bioassay described by Dautel et al., (1999), which yields highly reliable, reproducible results similar to tests involving human volunteers. In the moving-object bioassay (MOB), warmth and motion were used as attractants, stimulating the natural tick behaviour of clinging to a passing host under controlled laboratory conditions. A slowly rotating vertical drum was heated to body temperature (35-37°C) with the temperature carefully monitored using a remote infrared thermometer. A piece of filter paper (5 x 10 cm) was fixed at an elevated position on the drum, serving as the tick attachment site. Ticks are attracted to the warmth approach the drum on a horizontally positioned glass rod that ends directly in front of the drum at a distance where the tick cannot reach the drum surface by its forelegs. As the drum rotates, however, the elevated surface of the drum covered by filter paper passes periodically by and the tick is able to cling to that surface and transfer to the drum.

To test for tick repellency, blends of repellent compounds were applied to the filter paper using different doses 50 pi, 100 mI and 200 mI, corresponding to 1 mI/cm ² , 2 mI/cm ² and 4 mI/cm ² , respectively. The commercial repellents and ethanol control were tested at the highest doses (200mI). The filter paper was replaced between treatments. Observations were made of whether the tick approached and transferred to the drum and thereafter remained on the treated filter paper or dropped off. The duration of each step of tick behaviour was measured to reveal any subtle repellent effects. Behaviour observations were analysed using the Observer® software package (Nodus Information Technology, Wageningen, The Netherlands). Nymphs were gently placed at a line marked on the glass rod, approximately 8-12 mm from the tip. Standard experiments were performed at room temperature (20-22°C) and 40-70% RH. Experimenters maintained a reasonable distance (minimum 50cm) from the equipment during test runs. Ten nymphs were used for each treatment and the whole study repeated three times.

Statistical Analyses Frequency data such as the number of ticks walking versus not walking to the drum, or the number of ticks repelled versus not repelled are analysed by Fischer’s exact test. Metric data such as the times the ticks spent on treated versus untreated surface of the drum are analysed by one-way ANOVA followed by Tukey’s FISD test for unequal sample sizes in R (R Core Team, USA).

RESULTS Mosquito Repellence:

Essential Oil Olfactometry

Control assays resulted in 100% attraction over the test period in all cases. Overall there were significant differences between each assayed chemical at both 100% and 50% applied concentrations (F(6,63)=863.325, p=<0.001 & F(6,63)=79.695, p=<0.001 respectively). Of the four essential plant oil extracts tested, all produced a repellent effect significantly different from control responses at both 100% and 50% oil concentrations (Figure 1). Lavendula angustifolia and Rosmarinus officinalis were the least significantly different results (p= 0.11 & p=0.04 respectively) when compared to controls at 50% applied concentration. All other assays were significantly different to controls to the degree of p<0.001. Picea sitchensis was the most effective repellent oil from the 4 plant extracts tested, comparing favourably to DEET at concentrations of both 100% and 50% (p= 1.000 & p= 0.483). Ocimum suave was also an effective repellent, comparing favourably to DEET at 100% (p= 0.998), although being a less efficacious repellent at 50% (p=0.004). Citronella was comparable in terms of response to both P. sitchensis and 0. suave even at 50% (p= 0.249 & p= 1 .000).

Electroantennography

Of the 13 individual compounds tested for electrophysiological response, 10 produced a significant electrophysiological response as compared to hexane controls: (+)-Borneol, Bornyl acetate, 2- cyclohexen-1-ol, a-pinene, b-pinene, camphor, cineole, caryophyllene, eugenol, isoeugenol (F(13, 126)= 327.435, p<0.01 ). Of the other compounds assayed; a-terpineole, eugenyl acetate & acetic acid, none showed a response significantly greater than that of the control (p= 1 .000, p= 0.999 & p= 0.384 respectively) (Figure 2).

Single Chemical Olfactometry - Initial Characterisation

At 1 mg/cm ² applied concentration all compounds tested produced a repellent response as compared to controls (F(10,99)= 63.147, p<0.001 ). At 0.5mg/cm ² applied concentration significant repellent activity was again recorded for most compounds (F(10,99)= 44.652, p<0.001 ), with the exceptions of 2-cyclohexen- 1 -ol (p= 1.000) and b-pinene (p= 0.157). At 0.1 mg/cm ² applied concentrations only 6 of the chemicals tested produced repellent responses significantly different to controls; (+)-borneol, bornyl acetate, camphor, caryophyllene, eugenol and isoeugenol (F(10,99)= 154.125, p<0.001). Averaged responses can be seen in figure 3.

Four of these six chemicals (borneol, bornyl acetate, eugenol and isoeugenol) were selected for further assay to evaluate potential synergistic blends. As noted above, no significant variation in repellent activity was observed between different enantiomers or between individual and combinations of enantiomers, indicating that the observed repellent activity was not limited to any particular enantiomeric form. Therefore, whilst further assays were conducted using the specific enantiomers (-)-borneol and/or (-) bornyl acetate, it is expected that analogous results would be observed for compositions comprising (+)-borneol and/or (+)-bornyl acetate, or for mixtures of enantiomers. Single Chemical Olfactometry - Minimum Repellent Concentration

The repellence of test compounds coded B ((-)-Borneol), BA (Bornyl acetate), E (Eugenol), IG (Isoeugenol), C (camphor) were assessed, along with ethanol carrier and/or empty sample pad controls, using the BG-Cage test method. In initial tests, condensed cellulose sample pads were prepared comprising 0.5 mg/cm ² of each applied test compound. Tests for each test compound were conducted in triplicate, with the mean number of landings across all three tests being used to calculate the mean percentage mosquito trap catch relative to negative control. The mean number of landings for each component, percentage reduction compared to ethanol pad control and mean percentage catch rate are shown in Table 2 and Figure 4. Table 2: Mean number of landings for each component, % reduction from Ethanol pad and the mean % catch rate.

As shown in Table 2 above, tests conducted using sample pads containing 0.5 mg/cm ² IE or E or C resulted in a mean percentage mosquito trap catch of less than 35% relative to negative control. Thus, for the purposes of assessing synergy, the minimum repellent concentration (MRC) was calculated as 0.5 mg/cm ² for each of IE, E and C.

However, 0.5 mg/cm ² B or BA was not sufficient to provide the required mean percentage mosquito trap catch of 35% or less. Therefore, analogous tests were conducted using sample pads containing 0.7 mg/cm ² and 0.9 mg/cm ² of B or BA. The mean number of landings for each component, percentage reduction compared to empty pad control (ethanol control was not deemed necessary given the results presented in Table 2) and mean percentage catch rate are shown in Table 3 and Figure 5. Table 3: Mean number of landings for each component, % reduction from blank sample pad and the mean % catch rate.

As shown in Table 3 above, tests conducted using sample pads containing 0.7 mg/cm ² B or BA did not result in a mean percentage mosquito trap catch of 35% or less relative to negative control. However, increasing the concentration to 0.9 mg/cm ² did achieve the reach the required trap catch threshold of 35% or less. Thus, for the purposes of assessing synergy, the minimum repellent concentration (MRC) for B and BA was calculated as 0.9 mg/cm ² . Multi Chemical Blend - Four Component Synergy Testing

Potential synergistic interactions were assessed for blends comprising eugenol (E), isoeugenol (IE), (-)-borneol (B) and borneol acetate (BA). In particular, analogous BG-cage tests to those conducted for individual repellent compounds were conducted using sample pads containing the following applied blends:

Blend 1: 0.125 mg/cm ² E; 0.125 mg/cm ² IE; 0.225 mg/cm ² B;

0.225 mg/cm ² BA

Blend 2: 0.0625 mg/cm ² E; 0.0625 mg/cm ² IE; 0.1125 mg/cm ² B;

0.225 mg/cm ² BA;

Blend 3: 0.0313 mg/cm ² E; 0.0313 mg/cm ² IE; 0.0563 mg/cm ² B;

0.0563 mg/cm ² BA;

Blend 4: 0.0156 mg/cm ² E; 0.0156 mg/cm ² IE; 0.0281 mg/cm ² B;

0.0281 mg/cm ² BA;

(NB Blends 2 to 4 were prepared by 1/2, 1/4 or 1/8 dilution (in ethanol) of blend 1 , respectively). Tests for each blend were repeated 6 times, with the mean number of landings across all six tests being used to calculate the mean percentage mosquito trap catch relative to negative control. The mean number of landings for each blend, percentage reduction compared to ethanol pad control and mean percentage catch rate for each of are shown in Table 4 and Figure 6. Table 4: Mean number of landings for each repellent blend, % reduction from blank sample pad and the mean % catch rate. As shown in Table 4 above, tests conducted using blend 4, which contained the lowest concentration of applied repellent compounds, still resulted in a mean percentage mosquito trap catch of 35% or less relative to negative control. Thus, based on this data, the concentrations of E, IE, B and BA in blend 4 were considered to be the minimum concentration of each component in a mixture that produced the threshold repellent action. Therefore, these figures were considered alongside the MRC calculated for each individual repellent compound in order to calculate a Synergy Index (‘SI’) for the combination of E, IE, B. and BA as set out in Table 5.

Table 5: Synergy Index calculated for 4 component blend comprising borneol acetate, borneol, eugenol and isoeugenol.

The above data clearly demonstrates that the combination of borneol acetate, borneol, eugenol and isoeugenol represents a highly synergistic mosquito repellent formulation.

Multi Chemical Blend - Two Component Synergy Testing

To further characterise the synergistic interaction indicated above, potential synergistic interactions were assessed for blends comprising two components selected form eugenol (E), isoeugenol (IE), (-)-borneol (B) and borneol acetate (BA). In particular, analogous BG-cage tests to those conducted for individual repellent compounds and Four component blends were conducted using sample pads containing the following applied blends:

Blend 5: 0.0625 mg/cm ² E; 0.0625 mg/cm ² IE; Blend 6: 0.0625 mg/cm ² IE; 0.1125 mg/cm ² B; Blend 7: 0.0625 mg/cm ² IE; 0.1125 mg/cm ² BA; Blend 8: 0.0625 mg/cm ² E; 0.1125 mg/cm ² B; Blend 9: 0.0625 mg/cm ² E; 0.1125 mg/cm ² BA; Blend 10: 0.1125 mg/cm ² B; 0.1125 mg/cm ² E

Tests for each blend were repeated three times, with the mean number of landings across all three tests being used to calculate the mean percentage mosquito trap catch relative to negative control. The mean number of landings for each blend, percentage reduction compared to ethanol pad control and mean percentage catch rate for each of are shown in Table 6 and Figure 7.

Table 6: Mean number of landings for each two-component repellent blend, % reduction from blank sample pad and the mean % catch rate. As shown in Table 6 above, tests conducted using Blend 8, Blend 9 or Blend 10, each resulted in a mean percentage mosquito trap catch of 35% or less relative to negative control. Thus, based on this data, the concentrations of E and B; E and BA; and B and BA in Blends 8, 9 and 10 respectively were considered to be the minimum concentration of each component in a mixture that produced the threshold repellent action. Therefore, these figures were considered alongside the MRC calculated above for each individual repellent compound in order to calculate a Synergy Index (‘SI’) for two component combinations of E and B; E and BA; and B and BA as set out in Tables 7 to 9.

Table 7: Synergy Index calculated for 2 component blend comprising eugenol and borneol. Table 8: Synergy Index calculated for 2 component blend comprising eugenol and borneol acetate.

Table 9: Synergy Index calculated for 2 component blend comprising borneol and borneol acetate. The above data clearly demonstrates that the following combinations represent highly synergistic mosquito repellent formulations: eugenol and borneol; eugenol and borneol acetate; and borneol and borneol acetate.

In addition, and whilst it was not possible to calculate the synergy index for combinations of eugenol and isoeugenol or isoeugenol and borneol (as Blends 5 and 6 resulted in a mean percentage mosquito trap catch of slightly greater than 35% relative to negative control), this data still clearly demonstrates that compositions comprising each of these combinations retain potent mosquito repellent activity at significantly reduced total repellent compound concentration compared with that observed for compositions comprising a single repellent compound. In particular compositions comprising a combination 0.0625 mg/cm ² eugenol and 0.0625 mg/cm ² isoeugenol (i.e. total repellent compound = 0.125 mg/cm ² ) or a combination of 0.0625 mg/cm ² isoeugenol and 0.1125 mg/cm ² borneol (i.e. total repellent compound = 0.175 mg/cm ² ), were shown to provide broadly similar repellent activity to single component compositions comprising 0.5 mg/cm ² (eugenol or isoeugenol) or 0.9 mg/cm ² (borneol) repellent compound. Therefore, the fact that broadly equivalent activity was still observed upon a 4-fold reduction in repellent compound concentration at least suggests that combinations of eugenol and isoeugenol and/or isoeugenol and borneol also represent synergistic mosquito repellent formulations.

Tick Repellence:

Moving Object Bioassay

Blends comprising eugenol (E), isoeugenol (IE), (-)-borneol (B) and borneol acetate (BA) were also assessed to ascertain if the repellent activity exemplified against mosquitoes extends to other, non-insect, arthropods. In particular, moving object bioassays were conducted using the following blends, diluted 1 :1 in pure ethanol, and compared with negative controls (i.e. no treatment or ethanol) and to commercially available repellent formulations [DEET (Anti Brumm® Forte 30% w/w, Hermes Arzneimittel GmbH, Deutschland), Picaridin (Autan® Protection Plus 20% w/w, SC Johnson GmbH, Deutschland), PMD (Anti Brumm® Naturel, 20% w/w, Hermes Arzneimittel GmbH, Deutschland), and IR3535 (Jaico Muggenmelk Natural, 19.6% w/w, Omega pharma Nederland bv, The Netherlands)]:

Blend A: 75mg borneol, 75mg bornyl acetate, 75pl eugenol, and 75mI isoeugenol; Blend B: 75mg borneol, 75mg bornyl acetate, 75mI eugenol, 75mI isoeugenol, 75 mg camphor.

As shown in Tables 10 and 11 below, the moving object bioassay demonstrated that both Blend A and Blend B were 100% repellent as compared to blank controls (p<0.001), independent of dose. In contrast, the ethanol control had 3.3% repellency whilst the blank controls had negligible (0- 3.3%) repellency. Further, the activity of Blend A and Blend B compares well with the commercial repellents DEET (Anti Brumm Forte) and Picaridin (Autan Protection Plus), which were also 100% repellent compared to blank controls (p<0.001), while PMD (Anti Brumm Naturel) and IR3535 had 96.7 % and

93.3% repellency (p<0.001) respectively.

Table 10. Repellency and relative repellency (repellency corrected for the control) of the test products Blend A (200pl, 100 pi, 50 pi) and Blend B (200pl, 100 pi, 50 pi) and solvent control (ethanol) against Ixodes ricinus nymphs in the moving object bioassay.

Table 11. Repellency and relative repellency (repellency corrected for the control) of the test products Anti Brumm®Naturel (PMD) , Anti Brumm®Forte (DEET ), Autan® Protection Plus (Picaridin), IR3535 19.6% w/w ethanol solution against Ixodes ricinus nymphs in the moving object bioassay. Significances are indicated according to Bonferri corrected Fisher’s exact tests on the number of repelled ticks in test product runs and blank control runs. NS = not significant. Number of ticks = 30.

As shown in Table 12, in both the ethanol and blank controls, the majority of ticks moved to the drum; with 96.7-100% transferring to and remaining attached to the drum for the duration of the assay. However, the response of the ticks when exposed to Blend A or Blend B depended on the dose. At the lowest dose (50pl = 1 mI/cm ² ), all the ticks moved towards the drum treated with Blend A but only 6.7% transferred to the drum but these soon dropped of the drum. At the intermediate dose (1 OOmI = 2pl/cm ² ) 96.7% moved towards the drum but climbed onto the drum. At the highest dose of blend A (200mI = 4mI/ah ² ) 63.3% moved towards the drum (however, only a fraction of these (21.1%) transferred to the drum and all fell off). At the highest dose of Blend A approximately 37% did not move in the direction of the drum. For ticks exposed to the lowest dose of Blend B, 96.7% moved towards the drum, of which 6.9% transferred to the drum briefly as they soon fell off. At the intermediate dose of Blend B, 93.3% of the ticks moved towards the drum but did not climb onto the drum. At the highest dose of Blend B, 60% of the ticks moved towards the drum but did not climb onto the drum and 40% did not walk. The number of ticks that moved to the drum treated with PMD, DEET, Picaridin and IR3535 was 83.3% (ns), 63.3% (p <0.001), 70% (p<0.01) and 83.3% (ns), respectively. Of the ticks that had moved to the tip of the glass rod only a fraction transferred to the drum. The number of ticks that transferred to the drum treated with PMD, DEET, Picaridin and IR3535 was 16% (p<0.001), 10.5% (p <0.001), 33.3% (p<0.001) and 28% (p <0.001), respectively. The number of ticks that did not walk when exposed to these repellents was highest for Picardin (30%) and DEET (ca. 37%) and lowest for PMD and IR3535 (both were ca, 17%). Of the few ticks that transferred to the drum, all those exposed to DEET (p<0.01) and Picaridin (p<0.001) fell off. However, only 75% and 71.4% of those exposed to PMD (p<0.01) and IR3535 (p< 0.001) fell off, respectively.

Table 12 Number of Ixodes ricinus nymphs displaying certain behavioural steps in the moving object bioassay with the test products Blend A and Blend B and controls which include blanks and solvent control (ethanol) as well as commercial repellents Anti Brumm®Naturel (PMD)) , Anti Brumm®Forte (DEET),

5 Autan® Protection Plus (Picaridin), IR3535 19.6% w/w ethanol. The first two rows of each behavioural step show the number of ticks. The last row indicates the respective results of Bonferroni corrected Fisher’s exact tests between the number of ticks showing these behaviours and the control. NS = not significant

As shown in Table 13. the mean time taken for the nymphs to move from start point to the tip of the glass rod was 4.6-5.6 seconds for the blank controls and 6.9 seconds for the ethanol control. It took slightly longer for the highest doses of Blend A (8.8 sec) and Blend B (7.5 sec). At the lower doses the time taken ranged between 5.3 and 6 seconds. The mean time taken for the nymphs to move to the tip of the glass rod when exposed to the commercial repellents PMD, DEET, Picaridin and IR3535 was 7.3, 7.9, 10.7, 8.7 seconds, respectively. The time taken to move from the tip of the glass rod to the rotating drum ranged between 3.4 and 4.9 seconds for the blank controls and 5 seconds for the solvent control. Of the few ticks that transferred to the drum in the Blend A and 4 treatments the time taken at the lowest doses of blend A and Blend B was 38.9 and 4.8 sec, respectively. Of the few that transferred when exposed to the highest dose of Blend A the mean time taken was 5sec. Of the few ticks that transferred to the drum treated with PMD, DEET, Picardin and IR3535 the mean time taken was 10.4, 18.5, 8.5, 8.5 seconds, respectively. The time the ticks spent on the drum in blank controls ranged between 29.6 and 56.5 seconds and 47.8 seconds for the solvent control. The time spent by the ticks exposed to the lowest dose of Blend A and Blend B was 5 and 3 seconds, respectively. Surprisingly, of the few ticks that transferred to the drum treated with Blend A the mean time on the drum was 13.9 seconds but in the PMD, DEET, Picaridin and IR3535 treatments it was 11. 3.5, 9.2 and 40.3 seconds, respectively.

Summary

Mosquito Repellence

Initial olfactometry experiments clearly showed the high efficacies of both DEET and citronella oils as mosquito repellents at both 100 % and as low as 50 % applied concentrations. P. sitchensis extract did however compare favourably, particularly under tests completed using 100 % extracts. Aedes mosquitoes are known to be less influenced by a number of repellent products, including DEET, suggesting further that this genus has undergone different receptor evolution. Families members of pine have, however, been relatively poorly studied in terms of their repellent action. Its performance as a repellent outstripped all other tested extracts, and even outperformed citronella at 50 % applications. Considering the widespread usage of citronella, the potential for pine plant family extracts, and the compounds within, as effective means of mosquito repellence is therefore apparent.

Electroantennography of compounds known to be present in abundance in pine extracts revealed that a number of the compounds elicited electroantennographical and behavioural responses. Analysis of the effects of singular chemicals on mosquito behaviour revealed a number of repellent compounds, even at low doses of 0.1 mg/cm ² . Borneol, in particular elicited a strong repellent response from Ae. aegypti adults, repelling 90 % of mosquitoes at the lowest concentration. Interestingly, borneol has been seen to be a mild oviposition attractant under some circumstances. Other compounds identified as being efficacious repellents included borneol acetate, camphor, (-)-trans-caryophyllene, eugenol and isoeugenol.

Further, no significant variation in repellent activity was observed between different enantiomers, or between individual and combinations of enantiomers, indicating that repellent activity was not attributable to any particular enantiomeric form. Moreover, combination(s) comprising two or more compounds selected from borneol acetate, borneol, eugenol and isoeugenol have been shown to interact synergistically, and thus can be combined to provide enhanced efficacy insect repellant formulations.

Tick Repellence

Moving Object Bioassays clearly showed that the mosquito repellent formulations of the invention, i.e. combinations comprising two or more compounds selected from borneol acetate, borneol, eugenol and isoeugenol, were highly repellent to I. ricinus nymphs. The tested blends were equally or marginally more repellent than leading commercial products. At the highest dose, the tested blends appear to arrest tick movement and force retreat, suggesting the volatiles were being detected at some distance from the drum.

This study has, therefore, demonstrated the efficacy of novel blends of compounds against the most common tick species on the European continent; showing improved or equal potential as compared to the some of the most prevalent commercial repellents on the market to date.

References

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Abdullah, Z. S., & Butt, T. M. (2014). Preferences of the peripheral olfactory system of Western Flower Thrips, Frankliniella occidentalis towards stereoisomers of common plant volatiles. Chemoecology, 25(1 ), 47-51.

Dautel H., Kahl O., Siems K., Oppenrieder M., Milller-Kuhrt L. and Hilker M. (1999). A novel test system for detection of tick repellents. Ent. Exp. App 91. 431-441. Geier, M., Bosch, O. J., & Boeckh, J. (1999). Ammonia as an attractive component of host odour for the yellow fever mosquito, Aedes aegypti. Chemical Senses, 24(6), 647-653.

Maddrell SHP. Secretion by the Malpighian tubules of Rhodnius. The movement of ions and water. J Exp Biol. 1969;51:71-97.

Obermayr U, Rose A, Geier M. A novel test cage with an air ventilation system as an alternative to conventional cages for the efficacy testing of mosquito repellents. J Med Entomol. 2010 Nov;47(6):1116-22. doi: 10.1603/me10093. PMID: 21175061.

Table 14 Mosquito repellents (Commercial formulations of repellents used in this study not pure active)